Cardiac safety analysis of anti-HER2-targeted therapy in early breast cancer

To evaluate the cardiac safety of anti-HER2-targeted therapy for early breast cancer; to investigate whether trastuzumab combined with pertuzumab increases cardiac toxicity compared with trastuzumab; to evaluate the predictive value of high-sensitivity Troponin (hs-TnI) and QTc for the cardiotoxicity associated with anti-HER2 targeted therapy in early breast cancer. A total of 420 patients with early-stage HER2-positive breast cancer who received trastuzumab or trastuzumab combined with pertuzumab for more than half a year in Tianjin Medical University Cancer Hospital from January 2018 to February 2021 were included. Left ventricle ejection fraction (LVEF), hs-TnI values, and QTc were measured at baseline and 3, 6, 9, 12 months. Cardiotoxicity was defined as a decrease in LVEF of at least 10 percentage points from baseline on follow-up echocardiography. Cardiotoxicity developed in 67 of the 420 patients (15.9%) and all patients had LVEF above 50% before and after treatment. The incidence of cardiotoxicity in trastuzumab and trastuzumab combined with pertuzumab was 14.3% and 17.9%, respectively (P > 0.05). Logistic regression analysis showed that age, coronary heart disease, left chest wall radiotherapy, and anthracyclines sequential therapy were independent risk factors for cardiotoxicity (P < 0.05). The value of hs-TnI and QTc at the end of treatment (12th month) were selected for ROC curve prediction analysis and the area under the ROC curve was 0.724 and 0.713, respectively, which was significantly different from the area of 0.5 (P < 0.05). The decrease of LVEF in the study was mostly asymptomatic, from the heart safety point of view, the anti-HER2 targeted therapy for early breast cancer was well tolerated. Trastuzumab combined with pertuzumab did not significantly increase cardiotoxicity. However, subgroup analysis suggests that in the presence of coronary artery disease (CAD) and sequential treatment with anthracene, trastuzumab and pertuzumab may increase the cardiac burden compared with trastuzumab. Hs-TnI and QTc may be useful in monitoring and predicting cardiotoxicity associated with anti-HER2 targeted therapy for early breast cancer.


Methods
Study population. Patients diagnosed with early-stage HER2-positive breast cancer who received trastuzumab or trastuzumab combined with pertuzumab therapy for more than half a year from January 2018 to February 2021 in Tianjin Medical University Cancer Hospital were collected. The criteria for admission were as follows: Stage I-III breast cancer; HER2 positive; after breast cancer trastuzumab or trastuzumab combined with pertuzumab treatment for more than half a year; relatively complete clinicopathological data were obtained, including age of onset, menopausal status, tumor stage, histological grade, pathological type, hormone receptor status, Ki-67 expression level, surgical method, medication history, radiotherapy location, etc.; echo, cardiac biomarker and ECG were detected before targeted therapy, and the above tests were performed regularly every 3 months (± 2 weeks) on the day before treatment cycle; baseline LVEF ≥ 50%. Exclusion criteria: metastatic breast cancer; a history of congestive heart failure, uncontrolled ventricular arrhythmias or myocardial infarction; combined with other primary malignant tumors; incomplete clinical and pathological data or lost followup. Administration method of trastuzumab: a 3-week regimen with an initial load dose of 8 mg/kg, followed by 6 mg/kg for more than half a year; In weekly regimen, the initial loading dose was 4 mg/kg and then maintained at 2 mg/kg for more than half a year. Pertuzumab: A 3-week regimen with an initial dose of 840 mg and a maintenance dose of 420 mg for more than 6 months. Targeted therapies can be used in combination with chemotherapy.
Definition. Estrogen receptor (ER), progesterone receptor (PR) and HER2 were evaluated according to the scoring system recommended by the guidelines of the American Society of Clinical Oncology (ASCO) and the Association of American Pathologists (CAP). Breast Cancer staging is based on the American Joint Committee on Cancer (AJCC) Eighth Edition TNM Staging Guidelines. Body Mass Index (BMI) = weight (kg) ÷ height 2 (m). Corrected QT interval (QTc), QT interval refers to the time required for the whole process of ventricular depolarization and repolarization, from the beginning of QRS wave group to the end point of T wave. QTc = QT √ RR, RR represents heart rate 27 . The cardiotoxicity diagnosis in this study was LVEF decreased from baseline > 10% and to a value of < 50% 22  Data analysis. IBM SPSS Statistics 26.0 software was used for statistical data analysis, and GraphPad Prism 8.0 was used for plotting. Enumeration data were described by frequency and percentage, while measurement data were described by mean ± standard deviation. LVEF and QTc included in the analysis were normally distributed by K-S test. Paired T test was used for comparison before and after treatment, and independent sample T test was used for comparison between groups. The K-S test did not conform to the normal distribution of hs-TnI, Wilcoxon signed rank sum test was used for comparison before and after treatment, and Mann-Whitney U test was used for comparison between groups. Logistic regression model was used to analyze univariate and multivariate cardiotoxicity associated with anti-HER2 targeted therapy in breast cancer. Receiver operating curve (ROC) was used to predict the diagnostic value of hs-TNI and QTc for the cardiotoxicity associated with

Results
From January 2018 to February 2021, a total of 511 patients receiving anti-HER2-targeted therapy for breast cancer in Tianjin Medical University Cancer Hospital were collected and screened according to inclusion and exclusion criteria. Finally, 420 patients were included in the analysis (Fig. 1). The median follow-up time was 10 months (6-15 months). All patients included in the analysis were female, with 230 patients (54.8%) receiving trastuzumab and 190 patients (45.2%) receiving trastuzumab combined with pertuzumab. The median age was 52 years old (28-77 years old).The baseline clinical case characteristics of all patients were shown in Table 1.
Of the 420 patients included in the analysis, 67 had a decrease of more than 10% in LVEF, and the incidence of cardiotoxicity was 15.9%. Most of the patients had an asymptomatic decrease, and all the patients had an LVEF of more than 50% before and after treatment. No patients were discontinued due to heart-related adverse reactions, and there were no congestive heart failure or drug-related deaths. The incidence of cardiotoxicity in trastuzumab group (H group) and trastuzumab combined with pertuzumab (HP group) for breast cancer was 14.3% and 17.9%, respectively (P > 0.05). Single factor analysis showed that age, hypertension, coronary heart disease, left chest wall radiotherapy and anthracene sequential therapy were the risk factors for the cardiotoxicity related to anti-HER2-targeted therapy in early breast cancer (P < 0.  Table 2. Multivariate analysis of cardiotoxicity related to HER2-targeted therapy in early breast cancer showed that age, coronary heart disease, left chest wall radiotherapy, and anthracene sequential therapy were independent risk factors for cardiotoxicity (P < 0.05), (Fig. 2).
Comparison results of LVEF, hs-TnI and QTc between the non-cardiotoxic group and the cardiotoxic group at each time point are shown in Table 3.There was no significant difference in baseline LVEF and hs-TnI values between the two groups (P > 0.05), and there were significant differences in LVEF and hs-TnI values between the two groups 3, 6, 9 and 12 months (P < 0.05). There were significant differences in QTc between the two groups at baseline and 3, 6, 9 and 12 months (P < 0.05).
The incidence of cardiotoxicity in H group and HP group was 14.3% and 17.9%, respectively (P > 0.05), and there was no significant difference in the incidence of cardiac toxicity between the two groups. Next, we conducted subgroup analysis for different risk factors, and compared whether there were significant differences in LVEF, hs-TnI and QTc at each time point between H group and HP group, so as to analyze whether HP dual target therapy increased the influence on the heart under different risk factors compared with H single target therapy.

Subgroup analysis of LVEF in the two groups under different risk factors. In patients with CAD,
HP group showed a more significant overall decline in LVEF compared with H group, and significant differences in LVEF between the two groups were observed at the 9th and 12th months (P < 0.05); in sequential treatment with anthracene, the decrease of LVEF was more significant between the two groups, significant differences were observed between the two groups at 6, 9, and 12 months (P < 0.05); however, there was no significant difference in LVEF between the two groups at each time point under the condition of age ≥ 60 and left chest wall radiotherapy (P > 0.05), as seen in Table 4.

Subgroup analysis of hs-TnI in the two groups under different risk factors.
In patients with CAD, the overall trend of hs-TnI increase was more obvious in the HP group than in the H group, and there were significant differences in hs-TnI between the two groups at baseline and 3, 6, 9 and 12 months (P < 0.05); Dur- www.nature.com/scientificreports/ ing sequential treatment of anthracene, the increase of hs-TnI was more obvious in the HP group than in the H group, and there were significant differences in hs-TnI between the two groups at baseline and 3, 6, 9 and 12 months (P < 0.05); however, there was no significant difference between the two groups in the age of 60 years and the left chest wall radiotherapy (P > 0.05); as seen in Table 5.

Subgroup analysis of QTc in the two groups under different risk factors. In patients with CAD,
the QTc of HP group showed a more obvious prolongation trend than that of H group, and there were significant differences in QTC between the two groups at 6, 9 and 12 months (P < 0.05); QTc prolongation in HP group was more obvious than that in H group at 3, 6, 9 and 12 months after sequential treatment (P < 0.05); There was no significant difference in QTc at each time point in patients aged ≥ 60 years and receiving left chest wall radiotherapy (P > 0.05); as seen in Table 6.
Sensitivity and specificity of hs-TnI and QTc. According to the above research results, we found that the changes of hs-TNI and QTc were closely related to the changes of LVEF, and even showed significant changes prior to LVEF in some conditions. In order to further verify the sensitivity and specificity of hs-TnI and QTc on the cardiotoxicity related to anti-HER2 targeted therapy in breast cancer, we selected the parameters at the end of treatment, the 12th month, for ROC curve prediction analysis. The results showed that the areas under the ROC curve of hs-TnI and QTc were 0.724 and 0.713, respectively, which had a statistical difference compared with 0.5 (P < 0.05), indicating that hs-TnI and QTc have a certain predictive effect on the cardiotoxicity related to anti-HER2 targeted therapy in breast cancer (Table 7, Fig. 3).

Discussion
Trastuzumab and pertuzumab are monoclonal antibodies for anti-HER2 therapy. They have complementary effects in mechanism, and combined administration can achieve a more complete blocking effect on the HER2 pathway 4 . Some studies have shown that monoclonal antibodies are associated with an increased risk of cardiotoxicity, especially when combined with anthracyclines [17][18][19][20][21] . However, autophagy disorders in cardiomyocytes have been identified as another potential mechanism for cardiotoxicity caused by trastuzumab rather than pertuzumab recently 28 . This may provide further understanding and support for the mechanism by which the addition of pertuzumab in the course of anti-HER2-targeted therapy does not increase cardiac toxicity. In our study, a total of 420 patients with HER2-positive early-stage breast cancer received targeted therapy for more than half a year were eventually included to evaluate the cardiac safety of anti-HER2-targeted therapy. Studies have reported that trastuzumab related cardiotoxicity usually occurs in the median of 5-6 months 21,29,30 , therefore, we selected patients who received targeted therapy for more than 6 months for the study. The incidence of cardiotoxicity in this study was 15.9%, consistent with clinical reports of cardiotoxicity associated with anti-HER2 targeting (5-19%) [13][14][15][16] . All the patients had LVEF above 50% before and after treatment. During the follow-up, no patients were discontinued due to heart-related adverse reactions, and no patients had congestive heart failure or drug-related death. From the perspective of cardiac safety, anti-HER2-targeted therapy for breast cancer was well tolerated. Similar results were found in BERENICE, KRISTINE, TRAIN-2 studies, which all supported that trastuzumab combined with pertuzumab did not significantly increase cardiotoxicity [31][32][33] . In the NeoSphere and CLEOPATRA study, more than 90% of patients treated with trastuzumab and pertuzumab did not develop any grade of left ventricular dysfunction 34,35 . In addition, some studies have shown that trastuzumab targeted therapy is a safe treatment for most patients from a cardiac perspective with long-term follow-up [36][37][38] . In this study, the incidence of cardiotoxicity in H group and HP group was 14.3% and 17.9%, respectively (P > 0.05), suggesting the HP treatment has not increased in patients with cardiac toxicity observably, but in the anthracene sequential treatment group, the effects of trastuzumab and pertuzumab on cardiac function related indexes LVEF, hs-TnI and QTc were more obvious, suggesting that anthracene sequential therapy increased the effects of trastuzumab and pertuzumab on the heart. Table 7. ROC curve analysis of hs-TnI and QTc for the diagnosis of cardiotoxicity. The cutoff values are the parameter value corresponding to the maximum value obtained by subtracting sensitivity − (1-specificity), at this threshold, the value of the Yoden index is the highest, that means, the highest accuracy of disease diagnosis. CI confidence interval, ROC receiver-operator characteristic. www.nature.com/scientificreports/ Conclusions on the risk factors of cardiotoxicity associated with targeted therapy are mixed. In a German prospective study of 3940 patients and NSABP B-31, N9831 combined analysis, older age (> 50 years old) was a risk factor for trastuzumab-related cardiotoxicity, and another retrospective study showed that age of > 65 years old was a risk factor 39,40 . A multicenter study found that hypertension is a potential risk factor for trastuzumab-related cardiotoxicity 41 . Two other retrospective studies concluded that radiotherapy and obesity were high risk factors for trastuzumab-related cardiotoxicity 42,43 . Other studies have found that CAD, diabetes, dyslipidemia, smoking, anthracycline sequential therapy are anti-HER2 targeted therapy-related risk factors for cardiac toxicity [44][45][46][47] . In addition to age, CAD, left chest wall radiotherapy, and anthracene sequential therapy were found to be independent risk factors for cardiotoxicity associated with targeted therapy for breast cancer in this study. Therefore, when anti-HER2-targeted therapy is applied in clinical practice, the changes of LVEF, hs-TnI and QTc in patients with age ≥ 60 years old, CAD, left chest wall radiotherapy and sequential anthracene therapy should be closely watched.
LVEF is often used to diagnose cardiotoxicity in patients who may have cardiotoxicity in the course of antitumor therapy, but it is not sensitive to reflect the early heart injury. Echography is a more common method for LVEF assessment, which is more economical and convenient. MUGA also can be used for assessing LVEF but not every patient used this method. Cardiac biomarkers, as a new detection method, have been widely studied for early detection, evaluation and monitoring of cardiotoxicity. Results of a small multicenter cohort of 78 patients showed that elevated hs-TnI was associated with cardiotoxicity induced by anthracycline and trastuzumab, and suggested a close relationship between elevated hs-TnI and subsequent cardiac dysfunction 48 . In addition, three studies have suggested that hs-TnI can predict the cardiotoxicity associated with anthracycline and trastuzumab, but more evidence is needed to support the importance of evaluating drug-induced cardiac injury over time [49][50][51] . In contrast, a prospective study showed that hs-TnI levels were detectable in patients with early-stage breast cancer treated with anthracycline and trastuzumab during treatment, however, they were not associated with asymptomatic decreased LVEF and did not predict cardiotoxicity 52 . Based on the inconsistent conclusions reported in the above literatures, we collected the hs-TnI of the enrolled patients at each time point and conducted statistical analysis. The results showed that there was a significant correlation between hs-TnI and the cardiotoxicity related to anti-HER2 targeted therapy. There were significant differences in hs-TnI values between the cardiotoxic group and non-cardiotoxic group at 3, 6, 9 and 12 months (P < 0.05), suggesting that hs-TnI is closely related to the cardiotoxicity associated with anti-HER2-targeted therapy. Moreover, in the subgroup analysis, in the case of CAD, significant changes in LVEF were observed at 9 and 12 months after treatment in the HP group compared to the H group, while significant differences in hs-TnI were observed in the two groups from baseline. In the case of sequential anthracene treatment, the same is true. Both groups showed significant changes in hs-TnI before LVEF, suggesting that in the course of anti-HER2-targeted therapy, hs-TnI may be earlier than the changes in LVEF in some cases. Furthermore, the ROC curve prediction analysis also further supported the predictive value of hs-TnI in predicting the cardiotoxicity related to anti-HER2-targeted therapy in breast cancer. It is also suggested that early attention should be paid to the changes of hs-TnI in patients with CAD and sequential treatment of anthracene in the application of trastuzumab and pertuzumab in clinical practice.
Prolongation of QTc means delayed repolarization of the heart, which creates an electrophysiological environment conducive to the development of ventricular arrhythmias, most notably Torsade de Pointes (TDP), in severe cases, ventricular arrhythmias can be induced and even lead to sudden death 53 . Data have shown that tyrosinase inhibitors such as lapatinib cause QTC prolongation 54 . Monoclonal antibodies are considered to have less impact on QTc due to their larger molecules, which cannot directly enter the binding site of the drug channel, and their higher targeting specificity compared with small-molecule drugs 55 . Notably, anthracyclines that are often used in combination with monoclonal antibodies have been shown to be associated with prolonged QTc or other arrhythmias 56,57 . In this study, there were significant differences in QTc at each time point between the cardiotoxic group and the non-cardiotoxic group (P < 0.05), suggesting a significant correlation between QTc prolongation and cardiotoxicity associated with anti-HER2-targeted therapy. In addition, in the subgroup analysis, QTc of the two groups also showed significant changes earlier than LVEF in the case of coronary heart disease and sequential treatment of anthracene, suggesting that the change of QTc may be earlier than LVEF in some cases during anti-HER2-targeted therapy. Moreover, ROC curve prediction analysis also further verified the predictive value of QTc on the cardiotoxicity related to anti-HER2 targeted therapy in breast cancer.
This study holdings a few limitations. This is a retrospective study, possibly leading to selection bias. When using echocardiography to measure parameters, subjective differences among physicians are inevitable. Therefore, prospective studies should be conducted in the future to continue to expand the sample size and reduce the subjective differences among physicians.

Conclusion
In summary, the cardiac safety of anti-HER2-targeted therapy for early breast cancer was encouraging. Trastuzumab and pertuzumab did not significantly increase cardiotoxicity compared with trastuzumab. Age, CAD, left chest wall radiotherapy, and anthracene sequential therapy were independent risk factors for the cardiotoxicity associated with anti-HER2-targeted therapy in early breast cancer. Subgroup analysis suggests that in the presence of CAD and sequential treatment with anthracene, trastuzumab and pertuzumab may increase the cardiac burden compared with trastuzumab. Hs-TnI and QTc have certain predictive value for the cardiotoxicity related to anti-HER2 targeted therapy in breast cancer.

Data availability
The datasets used during the present study are available from the corresponding author upon reasonable request.